Bacterial vaginosis (BV) is the most prevalent cause of vaginitis among women of childbearing age (Holmes et al., Sexually Transmitted Diseases (1999)). The prevalence of BV in women varies depending on the population studied, but ranges from 4% to >50%. The occurrence of BV is associated with an increased risk of acquiring sexually transmitted diseases (STDs) including HIV (Martin et al., J Infect Dis 178:1053-1059 (1998); Schmid et al., Sex Transm Infect 76:3-4 (2000); Taha et al., AIDS 12:1699-1706 (1998); Sobel, Annu Rev Med 51:349-356 (2000); Schwebke, Curr Infect Dis Rep 2:14-17 (2000); Gupta et al., J Infect Dis 178:446-450 (1998); Hawes et al., J Infect Dis 174:1058-1063 (1996)), as well as preterm infertility (Sweet, Infect Dis Obstet Gynecol 8:184-190 (2000)), delivery of low birth weight infants (Hillier et al., N Engl J Med 333:1737-1742 (1995)), spontaneous abortion (Leitich et al., Am J Obstet Gynecol 189:139-147 (2003); Ralph et al., BMJ 319:220-223 (1999)), pelvic inflammatory disease, and various postoperative infections (Sobel, Annu Rev Med 51:349-356 (2000); Pybus & Onderdonk, Microbes Infect 1:285-292 (1999), and references therein).
The etiology of BV is complex and poorly understood (Sobel, Annu Rev Med 51:349-356 (2000)). It is commonly thought that BV results from replacement of the normal hydrogen peroxide-producing Lactobacillus sp. in the vagina with high numbers of Gardnerella vaginalis, Mycoplasma hominis, and Mobiluncus sp. (Pybus & Onderdonk, Microb. Ecol. Health Dis. 9:19-26 (1996); Workowski & Levine, Sexually Transmitted Diseases Treatment Guidelines, on the world wide web at cdc.gov/mmwr/preview/mmwrhtml/rr5106a.1.htm). This in turn leads to the development of an oxygen-depleted environment that facilitates the growth of strict anaerobes including Gram-negative species of Prevotella, Porphyromonas, Bacteroides, as well as Peptostreptococcus (Sobel, Annu. Rev. Med. 51:349-356 (2000)), and to higher cytokine levels in the cervix and vagina (Hay et al., Brit. Med. J. 308:295-298 (1994); McGregor et al., Am. J. Obstet. Gynecol. 170:1048-1060 (1994)). The cause(s) that trigger the depopulation of lactobacilli, changes in microbial community structure, and the overgrowth of other organisms are not fully understood. However, an increased incidence of BV is known to be positively correlated with multiple sex partners, the frequency of intercourse, and douching (Simpson et al., J. Pediatr. Adolesc. Gynecol. 17:249-255 (2004)). Since the development of BV has not been attributed to the presence or absence of any single bacterial taxon it is commonly diagnosed based on the existence of three of the following four symptoms: (a) thin homogeneous malodorous discharge; (b) vaginal pH fluid >4.5; (c) an amine odor from vaginal fluid when 10% KOH is added; and (d) the presence of “clue” cells (vaginal epithelial cells with adherent bacteria that obscure cell margins) (Amsel et al., Am. J. Med. 74:14-22 (1983)). Alternatively, the abundance of clue cells in Gram-stained vaginal smears can also be used as a means to diagnose BV (Nugent et al., J. Clin. Microbiol. 29:297-301 (1991)).
Curiously, up to 50% of women diagnosed with BV may not exhibit all or any of the classic symptoms (Sweet, Infect. Dis. Obstet. Gynecol. 8:184-190 (2000); Schmid, Int. J. Gynaecol. Obstet. 67 Suppl. 1:S17-S20 (1999); Schwebke, Int. J. Gynaecol. Obstet. 67 Suppl. 1:S21-S23 (1999)). Such asymptomatic women are diagnosed as having BV due to the absence of numerically abundant populations with cellular morphologies that resemble those of lactobacilli. The equating of absence of lactobacilli with the occurrence of BV, has gained wide acceptance despite the fact that numerous studies have shown that a significant fraction of women without BV symptoms lack appreciable numbers of lactobacilli. This conundrum has not been resolved, nor is it recognized by the Centers for Disease Control. For example, the CDC publication, Sexually Transmitted Diseases Treatment Guidelines 2002 states the following: “BV is a clinical syndrome resulting from replacement of the normal hydrogen peroxide producing Lactobacillus sp. in the vagina with high concentrations of anaerobic bacteria (e.g., Prevotella sp. and Mobiluncus sp.), G. vaginalis, and Mycoplasma hominis.” This guidance equates the absence of lactobacilli with the existence of BV, and asserts that even women without vaginal lactobacilli, without overt classical symptoms, have BV.
The pH of the vagina is thought to be a principle factor in governing the composition of the vaginal microbial community in reproductive age women. A low pH environment selects for various acid-tolerant bacterial populations that can colonize and reproduce under such conditions, while precluding those that cannot (Pybus & Onderdonk Microbes Infect. 1:285-292 (1999)). Shifts in the structure of the vaginal microbial community that result in replacement of lactobacilli as the numerically dominant species, regardless of the cause, are typically accompanied by an upward swing in the environmental pH. This in turn provides an opportunity for abnormal flora such as yeasts and various anaerobes and bacterial species associated with BV to proliferate. It seems that the production of lactic acid per se is important, but the particular species of Lactobacillus present is less so since it varies among women. It also has been postulated that the production of hydrogen peroxide also may be an important mechanism by which some species of Lactobacillus suppress the growth of bacterial species that might otherwise represent a health threat. For example, Eschenbach et al., (J. Clin. Microbiol. 27:251-256 (1989)) have reported that vaginal lactobacilli that produce hydrogen peroxide are present in 96% of healthy women, but they are found in only 6% of women with BV. Importantly, decreased numbers of lactobacilli are correlated with an increased risk of acquiring HIV and STDs (Cohen et al., AIDS 9:1093-1097 (1995); Sewankambo et al., Lancet 350:546-550 (1997); Taha et al., AIDS 12:1699-1706 (1998); Taha et al. J. Acquir Immune Defic. Syndr. Hum. Retrovirol. 20:52-59 (1999); Royce et al., J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 20:382-386 (1999); Martin et al., J. Infect. Dis. 178:1053-1059 (1998); Martin et al., J. Infect. Dis. 180:1863-1868 (1999)), and those that produce hydrogen peroxide have been shown to inactivate the HIV virus (Kiebanoff & Coombs, J. Exp. Med. 174:289-292 (1991), which might lower the risk of HIV acquisition. The possible role of hydrogen peroxide in preventing colonization of the vagina by pathogenic bacteria is appealing since hydrogen peroxide is known to be microbicidal. However, direct evidence for the excretion of hydrogen peroxide in vivo is lacking, and the apparent failure of hydrogen peroxide to similarly affect members of the vaginal microbiota is a paradox that has not been resolved.
Many investigators have studied the species composition of vaginal flora and paid heed to the species of Lactobacillus present. It is widely believed that the principal Lactobacillus species in the vagina of healthy women are Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri (Antonio et al., J. Infect. Dis. 180:1950-1956 (1999); Pavlova et al., J. Appl. Microbiol. 92:451-459 (2002)). There is, however, disagreement in the literature and various other species have been reported as members of normal vaginal flora. For example, Reid et al. (FEMS. Immunol. Med. Microbiol. 15:23-26 (1996)) sampled 100 healthy premenopausal women and cultivated the dominant aerobic or microaerophilic isolates of Lactobacillus from vaginal swab samples. Eight species were detected, the most common species being L. jensenii. The uncertainty regarding the actual species of Lactobacillus in the human vagina can in part be attributed to the difficulties of classifying lactobacilli on the basis of phenotypic criteria and the historical confusion surrounding the taxonomy of Lactobacillus. While investigators have focused attention on the role and importance of lactobacilli as members of the vaginal flora, the fact that between 10 and 42% of women lack appreciable numbers of lactobacilli (Eschenbach et al., Clin. Infect. Dis. 30:901-907 (2000); Hillier, AIDS Res. Hum. Retroviruses 14 Suppl 1: S17-S21 (1998); Larsen Monif, Clin. Infect. Dis. 32:e69-e77 (2001); Marrazzo et al., J. Infect. Dis. 185:1307-1313 (2002); Redondo-Lopez et al., Rev. Infect. Dis. 12:856-872 (1990)) has been nearly overlooked.
Prior efforts to characterize the vaginal flora have largely employed methods that are commonly used in clinical microbiology laboratories (Redondo-Lopez et al., Rev. Infect. Dis. 12:856-872 (1990), and references therein). These methods are inherently limited because they require cultivation of organisms on selective and nonselective media in the laboratory, after which they are classified into broad taxonomic groups based on phenotypic characters and microscopy. Slow growing, strictly anaerobic, or fastidious organisms may not be recovered by these methods. Others may have failed to grow because investigators are unaware of their inability to grow on selective media. Finally, the coarse classification methods used do not distinguish ecotypically distinct populations in samples. Traditional culture-dependent methods are tedious and labor intensive, and their use for the analysis of large numbers of samples is costly, permitting analysis of only small numbers of samples per study.
Recently, inventories of resident human bacterial flora done using cultivation-independent approaches based on analyses of 16S rRNA gene sequences have revealed a large degree of previously uncharacterized diversity even within well-studied and familiar microbial environments such as the human gingival crevice (Kroes et al., Microbiol. 96:14547-14552 (1999); Paster et al., J. Bacteriol. 183:3770-3783 (2001)), intestines (Favier et al., App. Environ. Microbiol. 68:219-226 (2002); Zoetendal et al., J. Nutr. 134:465-472 (2004)), inner ear (Frank et al., J. Clin. Microbiol. 41:295-303 (2003)), tongue (Kazor et al., J. Clin. Microbiol. 41:558-563 (2003)), and the esophagus (Pei et al., Proc. Natl. Acad. Sci. USA 101:4250-4255 (2004)).